An understanding of transmembrane transport phenomena at the molecular level is an important objective of contemporary biology. A major category of biological transport molecules is the transport ATPases, the most thoroughly studied of which can be subdivided into the FIFO ATPAse/ATP synthases of mitochondria, bacteria and chloroplasts, and the aspartyl-phosphoryl-enzyme intermediate family, which includes the Na+/K+-translocating ATPase of animal cell plasma membranes, the CA2+-translocating ATPase of sarcoplasmic reticulum, the H+/K+-translocating ATPase of gastric mucosa, and the H+-ATPase in the plasma membrane of Neurospora and yeast. In this laboratory we are working toward an understanding of the molecular mechanism of the Neurospora plasma membrane H+-ATPase anticipating that what is learned about the mechanism of this enzyme will contribute to our understanding of the molecular mechanisms of the other ATPases in the aspartyl-phosphoryl-enzyme intermediate family, and possibly transport ATPases in general. Ongoing research in this laboratory is attempting to elucidate several aspects of the chemistry of the H+-ATPase, including the identification of active site residues, determination of the primary amino acid sequence, and topological mapping of the orientation of the polypeptide chain in the membrane at several stages of the catalytic cycle. The studies proposed in this application will investigate the physical properties of this transport molecule. Specifically, we hope to 1) identify several detergents or other amphiphiles that will facilitate preparation of the ATPase, which is oligomeric as isolated, as stable, active, lipid-free, water soluble monomers or low molecular weight multimers thereof (protomers) and determine their molecular weights and thus monomer copy numbers, 2) characterize a variety of other physical properties of the ATPase monomers (protomers) obtained, including their shape and secondary structural composition in the presence and absence of ligands known to induce enzyme conformational changes, and their possible multiple domain structure, and 3) develop procedures for crystallizing the ATPase monomers (protomers) that are obtained. Our long term goal, outside the scope of this proposal, is an elucidation of the molecular structure of this ion pump.